To study the molecular mechanism of DNA replication, you incubated soluble E. co

Question

To study the molecular mechanism of DNA replication, you incubated soluble E. coli extracts with a mixture of dATP, dTTP, dGTP, and dCTP, all of which are labeled with the a-phosphate group. After a while, the incubation mixture was treated with trichloroacetic acid, which precipitate the DNA but not the nucleotide or very short oligonucleotides. The precipitate was collected and the extent of the radioactively label nucleotide incorporation into the DNA was determined.

a.) If any one of the four nucleotides is omitted from the incubation mixture, would you detect radioactivity in the precipitate? Explain

b.) Would 32P be incorporated into the DNA if only dTTP is labeled? Explain

c.) Would radioactivity be found in the precipitate if 32P labeled the b and g rather than the a phosphate of the deoxyribonucleotides? Explain.

Explanation / Answer

a. No

The DNA synthesis requires all 4 nucleides precussors. If anyone of the 4 nucleides precussors were omitted from the incubation mixture, incorporation of 32P into DNA is not posiible. Therefore, no radioactivity is found.

b. No

The 32P will be incorporated into the DNA if dTTP of 4 nucleides were labelled. Even though syntheis requires all 4 nucleides precussors , one radioactively labelled precursor is enough inorder for radiactivity to be observed in the newly syntheised DNA.

c. No

During DNA syntheis, only alpha-phosphate is retained, which is attached to 3'-OH group. Pi(pyrophosphate) is released. DNA polymerase catalysis the cleavage of pyrophosphate, which contains, the beta and gama phosphate groups; and forms a bond between 5'-alpha phosphate and 3'-OH group. Therefore, no radioactivity would be found.

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